Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHOD FOR IMPROVING THE IMMUNITY OF A COMPANION ANIMAL
FIELD
Embodiments of the invention relate to a method of improving the immunity of a
companion animal. More particularly, but not exclusively, embodiments of the
invention relate
to a method of administering to a companion animal a glucose anti-metabolite
to improve the
immunity of the companion animal.
BACKGROUND
Biological theories have correctly predicted the finding that a restriction of
caloric intake
by food deprivation slows down certain undesirable cellular processes in
laboratory animals,
many associated with aging and age-related diseases.
In particular, caloric restriction has been shown to consistently extend the
life span, delay
onset and slow tumor progression, and retard physiologic aging in many
systems. Indeed,
research spanning more than seventy years has shown that caloric restriction
is a nutritional
intervention that consistently extends longevity in animals. See Weindruch and
Walford, "The
Retardation of Aging and Disease by Dietary Restriction," Springfield, IL:
Charles C. Thomas
(1988); Yu, "Modulation of Aging Processes by Dietary Restriction," Boca
Raton: CRC Press
(1994); and Fishbein, "Biological Effects of Dietary Restriction," Springer,
New York (1991).
These effects of caloric restriction on life span and tumorigenesis have been
reported numerous
times since the early studies of McKay. See McKay et al., "The Effect of
Retarded Growth Upon
the Length of Lifespan and Upon Ultimate Body Size," J. Nutr., Vol. 10, pp. 63
¨ 79 (1935).
Indeed, over the past two decades, a resurgence of interest in caloric
restriction in gerontology
has led to the general acceptance that this dietary manipulation slows
physiologic aging in many
systems. See Weindruch and Walford, "The Retardation of Aging and Disease by
Dietary
Restriction," Springfield, IL: Charles C. Thomas (1988); Yu, "Modulation of
Aging Processes
by Dietary Restriction," Boca Raton: CRC Press (1994); and Fishbein,
"Biological Effects of
Dietary Restriction," Springer, New York (1991) and Masoro, E.J. "Overview of
Caloric
Restriction and Ageing," Mech. Aging Dev., Vol. 126, pp 913-922 (2005).
Reductions in fasting glucose and insulin levels and improvements in insulin
sensitivity
are readily measured biomarkers of caloric restriction. Calorically restricted
rodents exhibit
lower fasting glucose and insulin levels, and the peak glucose and insulin
levels reached during a
glucose challenge are reduced in those on caloric restriction. See Kalant et
al., "Effect of Diet
Restriction on Glucose Metabolism and Insulin Responsiveness and Aging Rats,"
Mech. Aging
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Dev., Vol. 46, pp. 89 ¨ 104 (1988). It is also known that hyperinsulinemia is
a risk factor
associated with several such disease processes, including heart disease and
diabetes (Balkau and
Eschwege, Diabetes Obes. Metab. 1 (Suppl. 1): S23 ¨ 31, 1999). Reduced insulin
levels and
body temperature are two of the most reliable indicators of this altered
metabolic profile (Masoro
et al., J. Gerontol. Biol. Sci. 47:B202-B208, 1992); Koizumi et al., J. Nutr.
117: 361 ¨367, 1987;
Lane et al., Proc. Nat. Acad. Sci. 93:4154 ¨4164, 1996).
Glucose anti-metabolites such as 2-deoxy-D-glucose are compounds related to
glucose.
However, due to structural differences from glucose such compounds block or
inhibit certain
aspects of carbohydrate metabolism and may therefore mimic the effects of
caloric restriction
(Rezek et al., J. Nutr. 106:143 ¨ 157, 1972). These anti-metabolites exert a
number of
physiological effects, including reduction of body weight, decrease in plasma
insulin levels,
reduction of body temperature, retardation of tumor formation and growth, and
elevation of
circulating glucocorticoid hormone concentrations. (For a review see Roth et
al., Ann. NY Acad.
Sci. 928:305 ¨ 315, 2001). These physiological effects result from inhibition
of carbohydrate
metabolism.
As such, use of glucose anti-metabolites as components for improving the
immunity in
mammals would be beneficial. The interaction between nutrition and the immune
response has
been an area of intense research over the past five decades. A bi-directional
interaction between
nutrition, immune response, and infectious disease was suggested by Scrimshaw
(Schrimshaw
NS, Taylor CE, Gordon JE. Interaction of nutrition and infection. Am J Med Sci
1959;237:367-
403) in the 1950's. Subsequently, it was recognized that malnourished
individuals were at risk
for infection. Follow up studies have demonstrated that deficiencies of most
micronutrients
result in impaired host defense. On the other hand, others have
demonstrated the
supplementation of certain nutrients beyond accepted requirements may improve
certain indices
of the immune response. Taken together, it is quite evident that nutrition is
able to impact
immunity. The dysregulation in immune function is a well-documented
consequence of aging.
This dysregulation can lead to an increased incidence of morbidity (illness)
and mortality (death).
Cell-mediated immunity, primarily T cells is clearly the component of the
immunity most
adversely affected with advancing age. For review, see Pawelec (Pawelec G,
Wagner W,
Adibzadeh M, Engel A. T cell immunosenescence in vitro and in vivo. Exp
Gerontol
1999;34:419-29). Age-related T cell immunity dysfunction has been implicated
as the cause of
many chronic degenerative diseases in elderly humans, including arthritis,
cancer, autoimmune
diseases, and increased susceptibility to infectious diseases. Many theories
exist that have been
put forth to try and explain the mechanism(s) responsible for this decline,
but no one theory can
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fully account for all the changes observed. Senior dogs have been reported to
show a decreased
immunity response compared to younger dogs. Older dogs also differ in the
makeup of their
immunity compared to younger dogs. Based on these observations, the aging
process results in a
dysregulation of the immune response in dogs too, as is similar with other
species and further
provides an opportunity for nutritional modulation in this, or related
species.
Thus, it would be beneficial to provide nutrition such as glucose anti-
metabolites having
physiological effects on the immunity, such as by altering the proliferative
ability of T and B
immune cells, and altering the relative distribution of immune cell
phenotypes, for example. It
would also be beneficial to provide a composition comprising such glucose anti-
metabolite
components. Accordingly, embodiments of the invention relate to such benefits.
SUMMARY
Embodiments herein relate to a method for improving immunity of a companion
animal.
The method can include, in one embodiment, administering to the companion
animal a glucose
anti-metabolite in an amount effective to improve the immunity of the
companion animal. The
glucose anti-metabolite can be selected from the group consisting of 2-deoxy-D-
glucose; 5-thio-
D-glucose; 3-0-methylglucose; 1,5-anhydro-D-glucitol; 2,5-anhydro-D-glucitol;
2,5-anhydro-D-
mannitol; mannoheptulose; and mixtures and combinations thereof. The companion
animal can
be a canine and a feline. In specific embodiments, the glucose anti-metabolite
can be
mannoheptulose. In specific embodiments, the composition can be a kibble,
which can be
nutritionally balanced and which can contain less than about 5%
mannoheptulose.
The method can include feeding from about 1 mg/kg to about 50 mg/kg
mannoheptulose
to the companion animal per day. The method can also include feeding from
about 1 mg to about
1000 mg mannoheptulose per day.
In certain embodiments, the method for improving immunity of a companion
animal
includes administering to the companion animal a glucose anti-metabolite in an
amount effective
to improve the immunity of the companion animal, wherein improving the
immunity includes
improving the ability of the immune system of the companion animal to respond
such that the
proliferative ability of T and B immune cells to respond to a stimulation
challenge is altered after
administration of the glucose anti-metabolite.
In certain embodiments, the method for improving immunity of a companion
animal
includes administering to the companion animal a glucose anti-metabolite in an
amount effective
to improve the immunity of the companion animal, wherein improving the
immunity comprises
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attenuating the decline of the immune system by attenuating the age associated
increase in
CD18+ cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1 is a graph of CD18+ immune cells for dog group 1.
FIG 2 is a graph of CD18+ immune cells for dog group 2.
FIG 3 is a graph of T cell mitogenic stimulation to ConA for dog group 1.
FIG 4 is a graph of T cell mitogenic stimulation to ConA for dog group 2.
FIG 5 is a graph of T cell mitogenic stimulation to PHA for dog group 1.
FIG 6 is a graph of T cell mitogenic stimulation to PHA for dog group 2.
FIG 7 is a graph of B cell mitogenic stimulation to PWM for dog group 1.
FIG 8 is a graph of B cell mitogenic stimulation to PWM for dog group 2.
FIG 9 is a graph of the average dog group 1 serum 8-0HdG level.
DETAILED DESCRIPTION
Definitions
As used herein, the articles including "the", "a", and "an", when used in a
claim or in the
specification, are understood to mean one or more of what is claimed or
described.
As used herein, the terms "include", "includes", and "including" are meant to
be non-
limiting.
As used herein, the term "plurality" means more than one.
As used herein, the terms "animal" or "pet" mean a domestic animal including,
but not
limited to domestic dogs (canines), cats (feline), horses, cows, ferrets,
rabbits, pigs, rats, mice,
gerbils, hamsters, horses, and the like. Domestic dogs and domestic cats are
particular examples
of pets and are referred to herein as "companion animals." It should be
understood that
throughout this disclosure when using the term animal, pet, or companion
animal, the animal,
pet, or companion animal is in a non-diseased state, unless otherwise stated.
As used herein, the terms "animal feed", "animal feed compositions", "animal
feed
kibble", "pet food", or "pet food composition" all mean a composition intended
for ingestion by
a pet. Pet foods can include, without limitation, nutritionally balanced
compositions suitable for
daily feed, as well as supplements and/or treats, which may or may not be
nutritionally balanced.
As used herein, the term "nutritionally balanced" means that a composition,
such as pet
food, has known required nutrients to sustain life in proper amounts and
proportions based on
recommendations of recognized authorities, including governmental agencies,
such as, but not
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limited to, Unites States Food and Drug Administration's Center for
Veterinarian Medicine, the
American Feed Control Officials Incorporated, in the field of pet nutrition,
except for the
additional need for water.
All oral doses of the invention are calculated per kilogram of body weight of
the mammal
5 unless otherwise indicated.
It should be understood that every maximum numerical limitation given
throughout this
specification includes every lower numerical limitation, as if such lower
numerical limitations
were expressly written herein. Every minimum numerical limitation given
throughout this
specification will include every higher numerical limitation, as if such
higher numerical
limitations were expressly written herein. Every numerical range given
throughout this
specification will include every narrower numerical range that falls within
such broader
numerical range, as if such narrower numerical ranges were all expressly
written herein.
All lists of items, such as, for example, lists of ingredients, are intended
to and should be
interpreted as Markush groups. Thus, all lists can be read and interpreted as
items "selected from
the group consisting of' ... list of items ... "and combinations and mixtures
thereof."
Referenced herein are trade names for components including various ingredients
utilized
in embodiments of the invention. The inventors herein do not intend to be
limited by materials
under a certain trade name. Equivalent materials (e.g., those obtained from a
different source
under a different name or reference number) to those referenced by trade name
may be
substituted and utilized in the descriptions herein.
The processes, methods, compositions, and apparatuses herein may comprise,
consist
essentially of, or consist of any of the features or embodiments as described
herein.
In the description of the various embodiments of the disclosure, various
embodiments or
individual features are disclosed. As will be apparent to the ordinarily
skilled practitioner, all
combinations of such embodiments and features are possible and can result in
preferred
executions of the disclosure. The scope of the claims should not be limited by
the preferred
embodiments set forth in the examples, but should be given the broadest
interpretation consistent
with the description as a whole. As
will also be apparent, all
combinations of the embodiments and features taught in the foregoing
disclosure are possible and
can result in preferred executions of the invention.
Embodiments of the invention
Embodiments of the invention relate to compositions comprising a glucose anti-
metabolite component selected from the group consisting of 2-deoxy-D-glucose;
5-thio-D-
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glucose; 3 -0-methylglucos e; 1,5 -anhydro-D-glucitol ; 2,5- anhydro-D-
glucitol; 2,5 -anhydro-D-
mannitol; mannoheptulose; and mixtures and combinations thereof. Without
intending to be
limited by theory, these components are accepted to be glucose anti-
metabolites. In another
embodiment, the components may be present in the recited compositions by
virtue of a
component of plant matter such as avocado, or other enriched source of
mannoheptulose such as
alfalfa, fig, primrose, and the like.
Embodiments of the invention also relate to a method of improving the immunity
of
mammals. Such methods relate to administering to the mammal a composition as
disclosed
herein, wherein the composition is effective at improving the immunity of the
mammal
regardless of the age of the mammal. In one specific embodiment, the method
relates to
improving the ability of the immune system to respond, also termed improving
immune response,
of a mammal, such as a companion animal, by administration of a composition
comprising a
glucose anti-metabolite. In one other specific embodiment, the method relates
to maintaining
and/or attenuating a decline of the immune system with aging of a mammal, such
as a companion
animal, by administration of a composition comprising a glucose anti-
metabolite.
Immunity can be divided into innate and adaptive immunity. The adaptive branch
of the
immune system is represented by cellular and humoral immunity and can be
defined by improved
T and B cell responses, which can be measured by assays such as, but not
limited to, tritiated
thymidine lymphoproliferative response. The innate branch of the immune system
is represented
by CD18+ immune cells, which can be measured by assays such as, but not
limited to, altered
relative and absolute percent of white blood cell populations as measured by
immunofluorescence.
Additionally, improving the immunity can include altering the proliferative
ability of T
and B immune cells, and altering the relative distribution of immune cell
phenotype, for example.
The mammals disclosed herein can include vertebrates and invertebrates, such
as for
example insects (e.g., the fruit fly) and/or nematodes (e.g., Caenorbabditis
elegans). Humans and
companion animals are disclosed herein.
The glucose anti-metabolite components as disclosed herein include 2-deoxy-D-
glucose,
5- thio-D-glucos e, 3- 0-methylgluc os e, anhydro sug ars including 1,5-
anhydro-D-glucitol, 2,5-
anhydro-D-glucitol, and 2,5-anhydro-D-mannitol, mannoheptulose, and mixtures
and
combinations thereof. Mannoheptulose is one particular glucose anti-
metabolite. In one
embodiment, mannoheptulose may be present in the recited compositions as a
component of
plant matter such as an avocado, avocado extract, avocado meal, avocado
concentrate or other
enriched source of mannoheptulose.
Non-limiting examples of enriched sources of
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mannoheptulose include alfalfa, fig, or primrose. The plant matter may include
the fruit, seed (or
pit), branches, leaves, or any other portion of the relevant plant or
combinations thereof.
Avocado (also commonly referred to as alligator pear, aguacate, or palta)
contains
unusually enriched sources of mannoheptulose, as well as related sugars and
other carbohydrates.
Avocado is a sub-tropical evergreen tree fruit, growing most successfully in
areas of California,
Florida, Hawaii, Guatemala, Mexico, the West Indies, South Africa, and Asia.
Species of avocado include, for example, Persea Americana and Persea nubigena,
including all cultivars within these illustrative species. Cultivars may
include 'Anaheim,'
`B aeon, " Creamhart, "Duke, "Fuerte, "Ganter, "Gwen, "Has s, "Jim, "Lula,
"Lyon,'
`Mexicola Grande,' Murrieta Green,'
Nabal,"Pinkerton,"Queen,"Puebla,"Reed,"Rincon,'
'Ryan,' Spinks,"Topa Topa,"Whitsell," Wurtz,' and `Zutano.' The fruit of the
avocado is
particularly preferred for use herein, which may contain the pit or wherein
the pit is removed or
at least partially removed. Fruit from Persea Americana is particularly
preferred for use herein,
as well as fruit from cultivars which produce larger fruits (e.g., about 12
ounces or more when
the fruit is mature), such as Anaheim, Creamhart, Fuerte, Hass, Lula, Lyon,
Murrieta Green,
Nabal, Queen, Puebla, Reed, Ryan and Spinks.
Plant matter from alfalfa, fig, or primrose is also reported to provide
relatively high levels
of mannoheptulose. Alfalfa is also referred to as Medicago sativa. Fig or
Ficus carica (including
Cluster fig or Sycamore fig, for example) may also be used, as well as
primrose or Primula
officinalis.
It has been discovered that particular levels of a component selected from 2-
deoxy-D-
glucos e ; 5 -thio-D-glucose; 3- 0-methylgluco se ; 1,5 -anhydro-D-glucitol ;
2,5 -anhydro-D-glucitol ;
2,5-anhydro-D-mannitol; mannoheptulose; and mixtures and combinations thereof
can be useful
herein. In particular, it has been found that relatively low levels, as well
as relatively high doses
of the component, while useful, may provide less than optimal efficacy for
desired purposes.
Dosage will depend upon the glucose anti-metabolite component used and will
vary depending
upon the size and condition of the mammal to which the glucose anti-metabolite
is to be
administered. Dosage in the range of about 0.0001 or about 0.001 grams/kg to
about 1 g/kg can
be beneficial in some embodiments. As used herein, when dosage in mg/kg is
used, the "mg"
refers to the level of the component, such as mannoheptulose, and "kg" refers
to kilograms of
body weight of the mammal, such as a dog or cat. Dosage at the lower range may
also be
appropriate when using 2-deoxy-D-glucose in large animals. Higher dosage,
particularly of
compounds such as 5-thio-D-glucose or mannitol, may also be readily tolerated.
In one
embodiment, the dosage of the component provided to a mammal on a daily basis
may be from
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about 0.1, 0.5, 1, 2, or 5 mg/kg to about 15, 20, 50, 100, 150, or 200 mg/kg,
and all combinations
of these ranges, wherein "mg" refers to the level of the component and "kg"
refers to kilograms
of body weight of the mammal. In one embodiment, the dosage to the mammal, on
a daily basis,
may be from about 1 mg/kg to about 15 mg/kg, from about 2 mg/kg to about 10
mg/kg, or from
about 2 mg/kg to about 5 mg/kg. In one embodiment, the dosage to the mammal,
on a daily
basis, may be from about 1 mg/kg to about 5 mg/kg, from about 1.5 mg/kg to
about 5 mg/kg,
from about 2 mg/kg to about 5 mg/kg, or about 2 mg/kg. In certain embodiments,
these amounts
may translate to compositions comprising less than about 5%, or less than
about 2%, or from
about 0.0001% to about 0.5%, or from about 0.1% to about 10%, or from about
0.1% to about
5%, of the component, all by weight of the composition. All ranges
therebetween are envisioned.
The level of component may be determined by one of ordinary skill in the art
based on a variety
of factors, for example, the form of the composition (e.g., whether a dry
composition, semi-moist
composition, wet composition, or supplement, or any other form or mixture
thereof). The
ordinarily skilled artisan will be able to utilize the preferred dosage and
determine the optimal
level of component within a given composition.
Similarly, the overall dosage amount of the component on a daily basis
provided to the
mammal may be provided. Such a daily dosage amount can be from about 0.1 mg
per day to
about 1000 mg per day. Such daily dosage amounts can be dependent on the size
of the mammal
consuming the composition. For example, in one embodiment, larger mammals may
consume
more than smaller mammals. Of course, that is consistent with the dosing
disclosed herein with
respect to a dosing amount per mass of the mammal. Thus, in one embodiment, as
the mammal
increases in size, more of the composition can be administered.
Accordingly, in one embodiment, such a daily dosage amount can correspond to
the
dosage on a daily basis per mass of the mammal, as described herein.
Specifically, daily dosage
amounts can range, in some embodiments, from about 0.1 mg per day to about
1000 mg per day,
or even more, depending on the size of the mammal and the daily dosage amounts
as described
above. In other embodiments, the daily dosage can be from about 1 mg per day
to about 500 mg
per day, or from about 1 mg per day to about 200 mg per day, or from about 1
mg per day to
about 100 mg per day, or from about 5 mg day per day to about 100 mg per day,
or from about 5
mg per day to about 80 mg per day, or from about 10 mg per day to about 50 mg
per day, or
about 40 mg per day. All ranges therebetween are also envisioned.
Similarly, wherein an extract or meal of plant matter is utilized in the
compositions
herein, levels of extract or meal may be dependent upon level of efficacious
component within
such extract or meal. Extracts and/or meals have been found herein which
comprise from about
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0.5% to about 99% of the glucose anti-metabolite component, alternatively from
about 0.5% to
about 75% of the glucose anti-metabolite component, alternatively from about
0.5% to about
50% of the glucose anti-metabolite component, alternatively, from about 0.5%
to about 25% of
the glucose anti-metabolite component, all by weight of the extract or meal.
Extracts and/or
meals have been found herein in which the glucose anti-metabolite component
may be from
about 0.5, 1, 2, 5, or 10% to about 15, 25, 50 or 75% by weight of the extract
and/or meal.
Accordingly, embodiments of the invention are directed to a composition that
is intended
for ingestion by a mammal. Compositions include foods intended to supply
necessary dietary
requirements, as well as treats (e.g., biscuits) or other food supplements.
Optionally, the
composition herein may be a dry composition (for example, kibble), semi-moist
composition,
wet composition, or any mixture thereof. Alternatively or additionally, the
composition is a
supplement, such as a gravy, drinking water, yogurt, powder, suspension, chew,
treat (e.g.,
biscuits) or any other delivery form.
Moreover, in one embodiment the composition can be nutritionally balanced,
such as a
pet food kibble. In another embodiment, the composition is not nutritionally
balanced, such as a
supplement, treat, or other delivery form for a pet.
The compositions used herein may optionally comprise one or more further
components.
Other components are beneficial for inclusion in the compositions used herein,
but are optional
for purposes of the invention. In one embodiment, the compositions may
comprise, on a dry
matter basis, from about 10% to about 90% crude protein, alternatively from
about 20% to about
50% crude protein, alternatively from about 20% to about 40% crude protein, by
weight of the
composition, or alternatively from about 20% to about 35% crude protein, by
weight of the
composition. The crude protein material may comprise vegetable-based proteins
such as
soybean, cereals (corn, wheat, etc), cottonseed, and peanut, or animal-based
proteins such as
casein, albumin, and meat protein. Non-limiting examples of meat protein
useful herein include
a protein source selected from the group consisting of beef, pork, lamb,
poultry, fish, and
mixtures thereof.
Furthermore, the compositions may comprise, on a dry matter basis, from about
5% to
about 40% fat, alternatively from about 10% to about 35% fat, by weight of the
composition.
Embodiments related to compositions of the invention may further comprise a
source of
carbohydrate. In one embodiment, the compositions may comprise from about 35%,
by weight
of the composition, up to about 50%, by weight of the composition,
carbohydrate source. In
other embodiments, the composition can comprise from about 35% to about 45%,
by weight of
the composition, or from about 40% to 50%, by weight of the composition,
carbohydrate source.
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Grains or cereals such as rice, corn, milo, sorghum, barley, wheat, and the
like are illustrative
sources of carbohydrate.
The compositions may also contain other materials such as, but not limited to,
dried whey
and other dairy by-products, beet pulp, cellulose, fiber, fish oil, flax,
vitamins, minerals, flavors,
5 antioxidants, and taurine.
The compositions may also contain other optional ingredients. Optional
ingredients can
include Probiotic components (Bifidobacteria and/or Lactobacillus) and
Prebiotic
(fructooligosaccharides) components. Examples and amounts of Probiotic
components and
Prebiotic components that can be included are disclosed in United States
Publication No.
10 2005/0158294, for example. Other optional ingredients that can be
included are omega 6 and
omega 3 fatty acids, carnitine, hexametaphosphate, glucosamine, chondroitin
sulfate, carotenoids
including beta carotene, vitamin E, and lutein, and those ingredients as shown
in Table 1 below.
Immunity
As disclosed herein, the compositions can be useful for improving the immunity
of
mammals, particularly companion animals. With respect to immunity, CD18 is a
pan-leukocyte
cell surface marker that has been shown to increase with age and during
inflammation (Valente.
Immunologic function in the elderly after injury--the neutrophil and innate
immunity; J Trauma.
2009; 67(5):968-74). Leukocytes play an important role in the pathogenesis of
tissue injury due
to inflammation. At the site of increased leukocyte¨endothelial cell
interaction, leukocytes in the
microcirculation interact with adhesion molecules on the endothelial cells,
which lead to rolling,
adhesion, and migration. The leukocytes subsequently release cytokines and
produce proteases
and superoxide radical species, which also participate in the inflammation
cascade. It is these
reactions of leukocytes that cause inflammatory tissue and endothelial cell
injury. The
expression of CD11b/CD18 (CR3), which is related to the phagocytic production
of reactive
oxygen intermediates (ROI) as described above, is up-regulated in monocytes
from older
subjects, in comparison to the younger subjects. As well, autoimmunity is due
in part to the
heterodimeric 132 integrin lymphocyte function-associated antigen-1 (LFA-1)
CD11a/CD18 over
expression. As a result, the increase in CD1 la/CD18 antigen density on
lymphocytes has been
discussed as an event in the mechanism leading to the decreased lymphocyte
proliferative
response in vitro and to other immunological dysfunctions reported in old
subjects (Chiricolo M,
Morini MC, Mancini R, Beltrandi E, Belletti D, Conte R. Cell adhesion
molecules CD1 la and
CD18 in blood monocytes in old age and the consequences for immunological
dysfunction.
Gerontology. 1995;41(4):227-34). As a result of all these previously reported
negative effects of
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CD18 over-expression seen with advancing age, it would be desirable to
mitigate/attenuate the
age-associated increase of CD18, which glucose anti-metabolites, and in
particular
mannoheptulose, are shown to do herein in companion animals, such as dogs, as
provided for
herein.
As shown in FIG 1, when analyzed by age, dogs that were fed a test diet
formulated to
deliver 2 milligrams mannoheptulose per kilogram body weight of the animal
showed a
significant attenuation in the increase of CD18+ immune cells when compared
against their diet
matched controls. The increase in CD18+ immune cells is thought to be an event
in the
mechanism leading to the decreased lymphocyte proliferative response in vitro
and to other
immunological dysfunctions reported in older subjects, therefore an
attenuation of this increase
would be considered beneficial immunologically to the mammal.
As shown in FIG 2, when analyzed by age, dogs that were fed the test diet with
mannoheptulose showed a trend for attenuation in the increase of CD18+ immune
cells when
compared against their diet matched controls. The increase in CD18+ immune
cells is thought to
be an event in the mechanism leading to the decreased lymphocyte proliferative
response in vitro
and to other immunological dysfunctions reported in older subjects, therefore
an attenuation of
this increase would be considered beneficial immunologically to the mammal.
The dogs of FIG 1, associated with a dog group 1, represent an older group of
dogs than
those of FIG 2, associated with a dog group 2, by about two years.
Concanavalin A (ConA) exhibits mitogenic activity, specifically with T-
lymphocytes
(Ruscetti and Chervenick 1975; Novogrodsky and Katchalski 1971; Perlmann et
al. 1970).
Phytohaemagglutinin (PHA), the lectin extract from the red kidney bean
(Phaseolus vulgaris),
contains potent, T-cell mitogenic activity (Hammerstrom, S. et al. (1982)
Proc. Nall. Acad. Sci.
USA 79, 1611-1615). Pokeweed mitogen (PWM), derived from Phytolacca Americana,
is a B
cell mitogen (Schreck et al., Annals of Clinical and Laboratory Science, Vol
12, Issue 6, 455-
462). Together these three mitogens are used to stimulate immune cells in
vitro in the
lymphocyte blastogenesis assay, a well accepted indicator of immune response
in vivo. Many
studies have shown this assay to be responsive to the age-associated decline
in immune system
response (Goldrosen et al., 1977 ¨ Journal of surgical oncology 9: 229-234). A
lower
proliferative capacity indicates the immune cells cannot become activated and
multiply as
efficiently (a key step in the process of mounting an effective immune
response). This decreased
proliferative capacity has been attributed to the increased morbidity and
mortality seen with
advancing age and hence is an important indicator of immune capacity.
Data from ConA, PHA, and PWM is shown in FIGs 3-8 for the two groups of dogs.
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For ConA, FIGs 3 and 4 indicate a benefit of mannoheptulose on T cell
proliferative
capability. All baseline values are similar (p>0.05), and either numerically,
statistically trending,
or statistically higher for dogs fed mannoheptulose-containing diets versus a
control diet. Taken
together, these observations support the conclusion that mannoheptulose is
beneficial on this
compartment of the immune cell population. T cell responsiveness declines with
age and is a
major contributing factor to the increased morbidity and mortality seen with
advancing age.
Therefore, reversing or mitigating this effect positively affects the health
and immune response in
dogs. Thus, FIGs 3 and 4 show that dogs which are fed a test diet containing
mannoheptulose
have either a statistical trend or a statistical improvement in T cell
mitogenic stimulation to
ConA. This increase indicates that the T cells of dogs fed a mannoheptulose
containing diet are
better able to respond to a challenge when compared to T cells from dogs fed a
control diet.
Similar to ConA, PHA responses shown in FIG 5 and 6 indicate a benefit of
mannoheptulose on the ability of the T cell compartment to respond to
mitogenic stimulation.
All baseline values are similar (p>0.05), and during the treatment period, all
PHA-stimulated T
cell data are either numerically, statistically trending, or statistically
higher for mannoheptulose
fed dogs versus controls. Taken together with the evidence seen with ConA,
these observations
support the conclusion that mannoheptulose is beneficial on the T cell
compartment of the
immune cell population. It is well known that T cell responsiveness declines
with age and is a
major contributing factor to the increased morbidity and mortality seen with
advancing age.
Therefore, reversing or mitigating this effect positively affects the immune
response, health, and
wellness of dogs. FIGs 5 and 6 above show equally that dogs which are fed a
test diet containing
mannoheptulose have either a statistical trend or a statistical improvement in
T cell mitogenic
stimulation to PHA. This increase indicates that the T cells of dogs fed a
mannoheptulose
containing diet are better able to respond to a challenge when compared to T
cells from dogs fed
a control diet.
The results shown in FIGs 7 and 8 indicate a benefit of feeding mannoheptulose
on the B
cell proliferative ability in dogs, shown as PWM in the figures. All baseline
data are statistically
similar and whenever a significant beneficial effect is seen, it is as a
result of feeding
mannoheptulose. B cell responsiveness is also known to decrease with age and
is also another
contributing factor to the increased morbidity and mortality seen with
advancing age. Therefore,
reversing or mitigating this effect positively affects the immune response,
health, and wellness of
dogs. FIGs 7 & 8 above show equally that dogs which are fed a test diet
containing
mannoheptulose have either a statistical trend or a statistical improvement in
B cell mitogenic
stimulation to PWM. This increase indicates that the B cells of dogs fed a
mannoheptulose
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containing diet are better able to respond to a challenge when compared to B
cells from dogs fed
a control diet.
8-0xo-2'-deoxyguanosine (8-0HdG) is an oxidized derivative of deoxyguanosine.
8-
OHdG is one of the major products of DNA oxidation. As part of the immune
response, T and B
cells must proliferate (become activated and divide). A key step to robust
proliferation is DNA
replication. If cells have damaged DNA or must repair DNA, this crucial step
can be hindered.
As shown in FIG 9, dogs that were fed a test diet containing mannoheptulose
had significantly
lower serum 8-0HdG compared to dogs fed a control diet. By lowering DNA damage
in the
mannoheptulose fed dogs, immune cell replication would be facilitated, helping
to enable a key
step in the activation cascade of the immune system.
As a summary, Table 1 is provided below. Table 1 shows the specific response
in the
first column. The second column is titled "General aging" and indicates what
happens to the
marker as aging occurs in many species. The third column relates to dogs and
matches the
second column of general aging of many species and thus indicates that without
mannoheptulose,
the signs of general aging in dogs will continue. However, in the fourth
column, the markers are
shown for aging when dogs consume a diet comprising mannoheptulose. As can be
seen, the T
cell proliferation and B cell proliferation responses increase when dogs
consume a diet
comprising mannoheptulose, while T cell proliferation and B cell proliferation
responses would
normally decrease with age. The same is true for CD18+ cells and DNA damage as
the
consumption of a diet comprising mannoheptulose will decrease these markers in
contract to
their typical increase with aging. Thus, a diet comprising mannoheptulose can
positively impact
the immune response, especially by way of attenuating the decline in the
immune response as the
dog ages and by improving the ability of the immune system to respond when
challenged.
Table 1
General aging Without MH With MH
T cell proliferation i i T
B cell proliferation i i T
% CD18+ cells T T i
DNA damage T T i
In another embodiment, an article of commerce is provided. The article of
commerce can
include a package. Any standard packaging that is used for deliver and sale of
the compositions
as disclosed herein can be used. The package can contain compositions
disclosed herein, such as
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compositions comprising mannoheptulose, or any other glucose anti-metabolite.
The
compositions can be nutritionally balanced pet food compositions. The package
can include
specific benefit statements written on the package. The specific benefit
statements can relate to
benefits that are provided to the consumer of the composition. The specific
benefit statements
can relate to improving the immunity when the composition is consumed. For
example, the
specific benefit statements can relate to improving the ability of the immune
system of a
companion animal to respond if the composition is administered to the
companion animal.
Another example includes specific benefit statements that can relate to
maintaining and/or
attenuating a decline of the immune system of an aging companion animal if the
composition is
administered to the aging companion animal. Thus, an article of commerce is
provided, wherein
the article of commerce comprises a package that contains a companion animal
composition,
wherein the package includes a benefit statement relating to improving the
immunity of the
companion animal if the companion animal consumes the companion animal
composition
contained within the package.
Examples
The following examples are provided to illustrate embodiments of the invention
and are
not intended to limit the scope thereof in any manner.
Preparation of Mannoheptulose-containing Avocado Meal:
Fresh avocados (Lula variety) were obtained from Fresh King Incorporated
(Homestead,
FL). The avocados were manually split open and the pits were removed and
discarded. The
remaining skin and pulp were ground through a Hobart Commercial Food
Preparation machine
(Serial No. 11-10410235) using a 12 1/4 sieve. The ground avocado was then
transferred to an
Edwards Freeze Drier (Super Modulyo Model, Crawely, Sussex, England). The
freeze drier was
set at ¨20 C for the first 24 hours, -5 C for the following 24 hours and 5
C for the final 72
hours. Upon removal from the freeze drier, the meal was ground to a powder
using a Straub
Grinding Mill (model 4E, Philadelphia, PA). The avocado meal was analyzed and
found to
contain about 10.35% mannoheptulose, by weight of the meal. It should be noted
that the
amount of mannoheptulose found in avocados varies with the particular strain
and state of
ripeness.
Preparation of Avocado Extract
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Avocado extract containing enhanced levels of mannoheptulose is prepared in
accordance
with the following optional process, and utilized in compositions of
embodiments of the
invention.
Whole avocado fruit (about 900 kilograms) is provided. The fruit is split and
the pits are
5 removed, either partially or wholly, providing about 225 kilograms of
pitted avocado halves.
The raw avocado is charged to a disintegrator, whereupon some agitation, water
(about 3000
kilograms) and CELLUBRIX (commercially available from Novozymes A/S) (about 1
liter) is
further charged. The mixture is further agitated and concurrently heated to
about 66 C. Upon
completion of the charge, further CELLUBRIX (about 1 liter) is added, and the
entire mixture is
10 held under agitation for about 12 hours at a controlled pH of about 5.5.
The temperature is then
further increased to about 80 C and then held for at least about 2 hours. The
resulting digested
plant mixture is then filtered at 80 C to provide the carbohydrate extract as
the filtrate. The
carbohydrate extract is then evaporated in a simplified recirculation system
at 80 C, under
vacuum, to provide the carbohydrate extract having from about 10% to about 20%
solids and a
15 pH of about 5.5. The extract is then further concentrated using a
refractance window dryer to
provide about 100 kilograms of the extract as a crystalline or powder (a yield
of about 11%
carbohydrate extract, based on the starting mass of the whole avocado fruit,
which is analyzed as
a yield from about 0.25% to about 4.5% mannoheptulose, based on the starting
mass of the whole
avocado fruit). It should be noted the amount of mannoheptulose found in
avocados varies with
the particular strain and state of ripeness of the fruit. The extract may be
used in the
compositions of embodiments of the invention.
Kibble compositions
Table 1 illustrates two kibble compositions having the following components at
the
approximate indicated amounts are prepared using methods which are standard in
the art,
including extrusion, and are fed to dogs and/or cats as a daily feed:
Table 1
Component Component Amount Component Amount
indicated as indicated as
Wt% Wt%
Extract of Avocado* 0.02 0.01
Chicken, Chicken By- 44 47
product Meal, Fish Meal,
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and Egg
Chicken Fat 8 6
Beet Pulp 2 3
Salts 2.5 2
Vitamins and Minerals** 1 1
Minors*** 3.5 4
Grains Remainder Remainder
(corn, sorghum, barley, rice,
wheat)
*Avocado may be substituted with other plant matter having enhanced
mannoheptulose content.
The incorporation of a mannoheptulose source likely replaces a similar amount
of a grain source
in the composition.
**Vitamins and Minerals may include: Vitamin E, beta-carotene, Vitamin A,
Ascorbic Acid,
Calcium Pantothenate, Biotin, Vitamin B12, Vitamin B1, Niacin, Vitamin B2,
Vitamin B6,
Vitamin D3, Vitamin D2, Folic Acid, Choline Chloride, Inositol, Calcium
Carbonate, Dicalcium
Phosphate, Potassium Chloride, Sodium Chloride, Zinc Oxide, Manganese Sulfate,
Copper
Sulfate, Manganous Oxide, Ferrous Sulfate, Potassium Iodide, Cobalt Carbonate.
***Minors may include: Fish oil, flax seed, flax meal, cellulose, flavors,
antioxidants, taurine,
yeast, carnitine, chondroitin sulfate, glucosamine, lutein, rosemary extract.
Administration
Eighty (n=80) Labrador Retrievers were randomized by age, gender, and
littermate to
receive either a complete and nutritionally balanced control diet that is
similar to Eukanuba
Senior Large Breed or an experimental diet that is identical to the control
diet except for the
inclusion of mannoheptulose as disclosed below. The dogs were split into two
study groups.
Study 1: A total of 39 older Labrador Retrievers are fed a nutritionally-
balanced
composition providing mannoheptulose at levels of 0 or about 2 mg/kg of body
weight of the
dog. Average age of the dogs (12 neutered males, 27 spayed females) at the
start of a 4-year
study is 6.7 years with a range of 5.1 to 8.2 years of age for the youngest
and oldest dog within
the cohort, respectively. The control composition is fed as a nutritionally-
balanced composition,
and it contains no mannoheptulose (0 mg/kg), avocado extract, avocado meal, or
avocado
concentrate. The test composition is the nutritionally-balanced control
composition formulated
with avocado extract, avocado meal, or avocado concentrate to provide
mannoheptulose at a dose
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of about 2 mg/kg body weight of the dog. Older dogs are fed one-half their
daily allotment of
food at 0730 and 1430 each day. Dogs were fed to maintain body weight and body
composition
score (BCS) within a 2-4 score range. If food adjustments were made, they were
made on a
quarterly basis. All dogs were fasted overnight and morning meals were
withheld until blood
collections could be conducted for all immune measurements. Water is provided
ad lib.
Study 2: A total of 41 younger Labrador Retrievers are fed a nutritionally-
balanced
composition providing mannoheptulose at levels of 0 or about 2 mg/kg of body
weight of the
dog. Average age of the dogs (12 neutered males, 29 spayed females) at the
start of the 36-
month feeding study is 4.0 years with a range of 2.0 to 6.1 years of age for
the youngest and
oldest dog within the cohort, respectively. The control composition is fed as
a nutritionally-
balanced composition (Eukanuba Senior Maintenance Formula), and it contains no
mannoheptulose (0 mg/kg), avocado extract, avocado meal, or avocado
concentrate. The test
composition is the nutritionally-balanced control composition formulated with
avocado extract,
avocado meal, or avocado concentrate to provide mannoheptulose at a dose of
about 2 mg/kg
body weight of the dog. Younger dogs are fed one-half their daily allotment of
food at 0730 and
1430 each day. Dogs were fed to maintain body weight and body composition
score (BCS)
within a 2-4 score range. If food adjustments were made, they were made on a
quarterly basis.
However, all dogs were fasted overnight and morning meals were withheld until
blood
collections could be conducted for all immune measurements. Water is provided
ad lib.
Methods
Blastogenesis Materials and Methods:
Canine whole blood was collected into heparin tubes and centrifuged at 600g
for 10
minutes. The buffy coat was transferred to a new sterile polypropylene tube
and diluted to 13m1
with PBS. This blood mixture was then layered onto 9m1 of room temperature
histopaque 1.077
and centrifuged for 30 minutes at 500g. The PBMC layer was removed from the
gradient and
washed with PBS. Remaining red blood cells were removed with ACK lyses buffer
(NH4CL-
155mM, EDTA-0.1mM, KHCO3-10mM, pH7.4) and PBMCs were washed again with PBS.
Cell count was determined on a Z2 Coulter Counter (Beckman Coulter).
Mitogens were purchased from Sigma and diluted in complete media (RPMI with
10%FBS and 1% PenStrep). Diluted Mitogens were added to the wells of a 96 well
tissue culture
plate at the following concentrations: ConcavalinA 2.5ug/ml, 5ug/m1 and
bug/mi. PHA
2.5ug/ml, lOug/m1 and 2Oug/ml. Pokeweed mitogen 0.25ug/ml, lug/ml and 5ug/ml.
PBMCs in
complete media were also added to each well at 2x10^5 cells per well. Total
volume of each
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well is 200u1. Plates were then incubated at 37 degrees in 5% CO2 for a total
of 72hrs. At 52hrs
(+/-2 hrs) the cells were pulsed with luCi per well of 3H-thymidine. Cells
were harvested and
radioactivity counted by liquid scintillation.
8-0HDG Assay:
Assay was run using canine serum samples separated from whole blood. Amount of
8-
OHDG is quantified using a commercially available ELISA kit from Oxis Health
Products,
catalog #21026.
Bring all reagents and samples to room temperature before use.
1. Reconstitute the Primary Antibody with the Primary Antibody Dilution
Buffer.
2. Add 50 mL of sample or standard per well. To prevent edge effects, do not
use
outermost rows (Rows A and H).
3. Add 50 mL of reconstituted Primary Antibody to all wells except Blank. Seal
plate
tightly with Plate Seal. Shake plate from side to side to mix fully. Incubate
at 37 C for 1 hour.
4. Pour off contents of plate. Pipette 250 mL diluted Washing Buffer into each
well.
Wash thoroughly by agitation, dispose of Washing Buffer. Invert plate and blot
against clean
paper towel to remove any remaining washing buffer. Repeat wash twice.
5. Reconstitute the Secondary Antibody with the Secondary Antibody Dilution
Buffer.
6. Add 100 mL of reconstituted Secondary Antibody per well. Seal plate tightly
with
Plate Seal. Shake plate from side to side to mix fully. Incubate at 37 C for 1
hour.
7. Dilute the Chromogen with 100 volumes of Chromogen Dilution Buffer.
8. Repeat step 4.
9. Add 100 mL of the diluted Chromogen per well. Shake plate from side to side
to mix
fully. Incubate at room temperature in the dark for 15 minutes.
10. Add 100 mL of the Stop Solution, mix, wait 3 minutes and read the
absorbance at 450
nm.
Flow Cytometry ¨ CD18 Method:
Canine whole blood was collected into EDTA tubes and centrifuged at 400g for
30
minutes. The buffy coat was transferred to a new sterile polypropylene tube
and diluted with 2m1
PBS. Red blood cells were lysed by adding 5m1 H20 for one minute and stopping
with 5m1 2X
cold PBS. Cells were centrifuged at 300g for 10 mm to pellet. The lysis step
was repeated until
a white cell pellet was obtained. Cells were then resuspended in 0.8m1 FACS
Wash buffer (PBS
with 1% MS and 0.02%NaN3). 100u1 of cell suspension was aliquoted to each FACS
tube for
staining.
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Staining antibodies were purchased from Serotec. Primary antibody: mouse anti-
dog
CD18 (MCA1780) used at 1:10 dilution. Secondary antibody: goat anti-mouse IgG
(H&L) FITC
(STAR117F) used at 1:50 dilution. Negative control antibody: mouse IgG1
FITC/Rat IgG2a
RPE (DC050) used at 1:10 dilution. Cells were incubated with each antibody for
30 mm on ice,
washed with FACS Wash Buffer and then fixed with 4% Formalin before analysis.
Cell
populations were analyzed on a FACScan Flow Cytometer (Becton Dickinson) using
CellQuest
Pro Software.
The dimensions and values disclosed herein are not to be understood as being
strictly
limited to the exact numerical values recited. Instead, unless otherwise
specified, each such
dimension is intended to mean both the recited value and a functionally
equivalent range
surrounding that value. For example, a dimension disclosed as "40 mm" is
intended to mean
"about 40 mm."
Every document cited herein, including any cross referenced or related patent
or
application, is hereby incorporated herein by reference in its entirety unless
expressly excluded or
otherwise limited. The citation of any document is not an admission that it is
prior art with
respect to any invention disclosed or claimed herein or that it alone, or in
any combination with
any other reference or references, teaches, suggests or discloses any such
invention. Further, to
the extent that any meaning or definition of a term in this document conflicts
with any meaning
or definition of the same term in a document incorporated by reference, the
meaning or definition
assigned to that term in this document shall govern.
While particular embodiments of the invention have been illustrated and
described, it
would be obvious to those skilled in the art that various other changes and
modifications can be
made without departing from the spirit and scope of the invention. It is
therefore intended to
cover in the appended claims all such changes and modifications that are
within the scope of this
invention.
